Troubleshooting Cell Culture Media for Bioprocessing

Introduction

Cell culture media, including basal media and feeds, are key elements impacting the performance of bioprocesses. Advancements in cellular and metabolic understanding, coupled with high throughput applications, have led to evolved approaches in medium development and optimization resulting in innovative cell culture media with desired characteristics to meet specific needs.

However, challenges associated with the use of cell culture media still exist.  Examples include manufacturability, subpar stability, inconsistent performance and/or underperformance in terms of productivity and quality attributes.  In some cases, troubleshooting efforts can be guided by learned know-how but in other circumstances a systematic approach is necessary to identify the root cause.  In this Ask the Expert session, we invite you to participate in discussions on experience and lessons learned around cell culture media troubleshooting. Please submit your questions about cell culture media for bioprocessing.

This session is no longer taking submissions. To be notified of new Ask the Expert sessions, join our mailing list.

Question 1

What stability issues are different for powder media compared to liquid media? Are there any differences in compositions between them?

0 0 0 Total: 0 Powder media are typically more stable than liquid media and therefore tend to have a longer shelf life. The salts in liquid media exist in ionized forms and are subject to reaction and degradation. Another common stability issue for liquid media is precipitation of components that are less soluble or … Continued

Question 2

Is there known correlation between media composition and glycosylation of proteins?

0 0 0 Total: 0 There are medium components known for impacting protein glycosylation through different mechanisms. For example, manganese serves as a cofactor in the glycosylation pathway and can improve glycosylation; galactose impacts glycan precursors and may improve glycosylation; high glutamine in medium formulation can cause less glycosylation due to ammonia generation.

Question 3

Are there media with carbon source different from glucose?

0 0 0 Total: 0 Yes, there are media with alternate carbon sources. For example, galactose, which has a lower metabolizing rate than glucose, may be supplemented into cell culture media to remedy the issue of high lactate generation in certain cell culture situations. Disaccharides are also commonly used in culture media for non-mammalian cells.

Question 4

What media components can reduce apoptosis and increase cell viability beyond critical density?

0 0 0 Total: 0 Apoptosis is typically caused by nutrient depletion and/or metabolite accumulation. Nutrient depletion can be prevented by proper feeding strategies. Metabolite accumulation is less of concern in a perfusion process but is often challenging to tackle in a fed-batch process. Media optimization approaches to control the accumulation of metabolites may include: … Continued

Question 5

Is it always important to supplement media with depleted components?

0 0 0 Total: 0 Nutrient depletion causes cell apoptosis which can impact productivity levels and/or product quality therefore it is important to supplement media with depleted components. In order to achieve the best performance, a metabolically balanced feed formulation and an optimal feeding schedule should be designed and applied.

Question 6

Is chemically defined media equivalent substitution for yeastolate and other non-defined components?

0 0 0 Total: 0 For recombinant protein and monoclonal antibody production, there are commercially available chemically defined cell culture media with equivalent or better performance than non-defined media. For vaccine production applications, media with non-defined components are still widely used.

Question 7

Which serum free media is suitable for optimization of CHO and BHK cells (Cell Culture) producing recombinant blood clotting factors.

0 0 0 Total: 0 There are commercially available serum free media suitable for the optimization of recombinant blood factor expression in CHO or BHK cells. Depending on the cell type and specific molecule of interest, the media may need to be supplemented with other components (e.g., growth factors, cell protection agents, and product dependent … Continued

Question 8

Which composition of fatty acids is suitable for CHO and MDCK? Is cholesterol essential to grow cell line mentioned above in fed-batch culture?

0 0 0 Total: 0 Mammalian cells can synthesize fatty acids however they cannot introduce double bonds into fatty acids beyond C9. Therefore, unsaturated fatty acids, such as linoleic acid and linolenic acid, are beneficial for mammalian cell culture. Cholesterol is not essential for mammalian cells except for auxotrophic mutants (e.g.,NS0 cells).

Question 9

We are seeing a rise in ammonia in one of our CHO cultures prior to harvest. It isn’t entirely predictable so we are having trouble troubleshooting. Any suggestions?

0 0 0 Total: 0 The concentration of glutamine and/or asparagine in your cell culture media should be assessed and limited since both amino acids can be deaminated leading to ammonia as the byproduct. During late stationary phase of mammalian cell culture, cells may utilize alanine to generate pyruvate and this process also produces ammonia. … Continued

Question 10

Can you explain how cell metabolism characteristics can be used to optimize media in CHO cells?

0 0 0 Total: 0 There are several ways of utilizing cell metabolism characteristics for medium optimization. Firstly, the metabolism rate of key medium components such as amino acids and vitamins can easily be determined by spent media analysis. The metabolic profiles can then be used for design of stoichiometrically balanced medium formulations. Since metabolic … Continued

Question 11

What techniques can you suggest for improving media solubility for media that is difficult to get into solution?

0 0 0 Total: 0 There are approaches, such as alternating pH (either acidic or basic) and temperature (mostly warm), that can accelerate/facilitate solubilizing media compounds. However, they don’t necessarily improve solubility (maximum concentration in a saturated solution)issues. Therefore, it is important to also evaluate the composition of your media formulation to assess whether there … Continued

Question 12

Which composition of fatty acids is suitable for CHO and MDCK? Is cholesterol essential to grow cell line mentioned above in fed-batch culture?

0 0 0 Total: 0 Mammalian cells can synthesize fatty acids however they cannot introduce double bonds into fatty acids beyond C9. Therefore, unsaturated fatty acids, such as linoleic acid and linolenic acid, are beneficial for mammalian cell culture. Cholesterol is not essential for mammalian cells except for auxotrophic mutants (e.g. NS0).